The parent grant for this project is R37 AI4102, Mitochondrial DNA of Normal and mutant Trypanosomes. This project builds on studies in the parent grant that identified proteins and genes for some of the components of the editing machinery (editosome) in Trypanosoma brucei. The parent grant provides significant biological insights for this organism, comparative analysis of the editosome in different kinetoplastid species will provide an exceptional opportunity to assess the physiology and evolution of editosome composition and function. This analysis will not only have practical value in the identification of genes not called by predictive algorithms but provides an invaluable additional level of insight on the molecular mechanisms of RNA editing in kinetoplastid parasites. In this proposal we will establish the composition of the editosome, determine the critical major kinetoplastid parasites of humans; T. brucei, T. cruzi, and Leishmania major. This will be accomplished by exploiting the information from T. brucei for rapid identification of editosome genes and proteins in the other two major kinetoplastid parasites. This project will employ a combination of in silico analyses (which have already identified many candidate genes in T. brucei) and direct assays that exploit affinity tag technology combined with tandem mass spectroscopy. Furthermore, the morphology of catalytically active editosomes will be determined by electron microscopy (EM), since the available data indicate substantial structural differences between Trypanosoma and Leishmania species. The overall goal of this proposal is to identify conserved and divergent structural and functional features of the editosome in these three species. These analyses will be useful for understanding the function of the editosome and its components and also for development of drugs that could be effective for all three species.